The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. As an effort to understand the physiological functions and the underlying molecular mechanisms of the N-end rule pathway, we began the biochemical and genetic dissection of this pathway in mice. We have shown the functions of N-terminal asparagine-specific deamidation in socially conditioned behavior, of N-terminal arginylation in cardiovascular development, and of N-terminal cysteine oxidation as an oxygen sensor. Mammalian UBR1/E3calpha is the first identified ubiquitin ligase (E3) of the ubiquitin system, and has been known as the only E3 that recognizes type 1 and type 2 N-terminal destabilizing residues of proteins. For the last two decades, the in vitro biochemical studies have suggested numerous biological functions of UBR1, including neuronal cell differentiation, amphibian limb regeneration, apoptosis, muscle wasting, and the degradation of various proteins (Sindbis virus RNA polymerase, HIV integrase, the Listeria monocytogenes p60, RGS4 and RGS16, and the encephalomyocarditis virus 3C protease). Surprisingly, however, mice lacking UBR1 were apparently normal except for the subtle defects in muscle protein degradation and fat metabolism. We hypothesize that the substrate recognition in the mammalian N-end rule pathway is mediated by the cooperative activity of a set of distinct E3s. Indeed we identified novel UBRl-like E3s termed UBR2 and UBR3, and further hypothesize that UBR2 and/or UBR3 are the E3(s) that may cooperatively function with UBRI. As a preliminary effort to address these issues, we have constructed UBR2 -/-, UBR3 -/-, and UBR1-/-UBR2 -/- and UBR1-/-UBR3 -/- mice, and found that UBR2-1oss caused male-specific infertility and female-specific lethality. Thus, we hypothesize that UBR2 is essential for spermatogenesis. This proposal focuses on the following aims: (1) To characterize the biochemical properties of UBR2 and UBR3 as candidate E3s that may function cooperatively with UBR1 in the N-end rule pathway, (2) To characterize UBR2 -/- mice to assess the in vivo function of UBR2 in spermatogenesis, (3) To identify the UBR2-interacting proteins or UBR2-dependent molecular circuits underlying the UBR2- dependent spermatogenesis.